J. Phys. Chem. C 2007, 111, 8655-8660
8655
DNA-Hemoglobin-Multiwalls Carbon Nanotube Hybrid Material with Sandwich Structure: Preparation, Characterization, and Application in Bioelectrochemistry Qian Zhang,†,‡ Ling Zhang,†,‡ and Jinghong Li*,† Department of Chemistry, Key Lab of Bioorganic Phosphorus Chemistry & Chemical Biology, Tsinghua UniVersity, Beijing 100084, China, and Department of Chemistry, UniVersity of Science and Technology of China, Hefei 230026, China ReceiVed: February 25, 2007; In Final Form: April 3, 2007
A novel multiwall carbon nanotubes (MWNTs)-based hybrid material with sandwich structure (DNA-HbMWNTs) was fabricated by alternative electrostatic assembly of hemoglobin (Hb) and DNA on MWNTs. TEM showed that such a nanocomposite behaved as an obvious core-shell structure. SEM proved the wellpreserved 3-D structure of DNA-Hb-MWNTs assembled on an electrode. UV-vis and FTIR spectroscopy were used to monitor the assembly procession and also demonstrated that Hb had been sandwiched into DNA and MWNTs without denaturation. A pair of stable and well-defined redox peaks of Hb with a formal potential of about -0.298 V (vs Ag/AgCl) in a pH 6.0 phosphate buffer solution (PBS) were obtained at the DNA-Hb-MWNTs nanocomposite film-modified glassy carbon (GC) electrode (DNA-Hb-MWNT/GC electrode). Compared with the Hb-MWNTs/GC electrode, the DNA-Hb-MWNTs/GC electrode exhibited enhanced faradic current response, and the portion of the electroactive proteins had been greatly improved. Furthermore, the modified electrode also displayed good sensitivity, wide linear range, and long-term stability to the detection of hydrogen peroxide. Such an organized multicomponent biosensor platform may find wide potential applications in biosensors, biocatalysis, biomedical devices, and bioelectronics.
1. Introduction Direct electron transfer (DET) of metalloproteins is an important research subject due to its application in the areas of biochemical and biophysical sciences.1-4 The DET between metalloproteins and electrodes can not only be applied to study enzyme-catalyzed reactions in biological systems, but also establish a foundation for fabricating mediator-free biosensors, biomedical devices, and enzymatic bioreactors.5 However, it is usually difficult for metalloproteins to display DET at conventional electrode surfaces due to the deeply embedded redoxactive center, unfavorable orientation, and denaturation of proteins. Therefore, suitable materials and methods for protein immobilization on the electrode surface are important for obtaining their direct electrochemical reaction and keeping their bioactivities. Carbon nanotubes (CNTs) represent an important group of nanomaterials with unique properties such as the high electrochemically accessible area of porous tubes, good electronic conductance, and strong mechanical property, which make them very promising in fields of electrochemical application, especially in bioelectrochemistry.6-8 So far, several metalloproteins have been successfully immobilized on the surface of CNTs to fabricate functional CNTs-proteins bio-nanocomposites and construct mediate-free biosensors.9-11 In addition, CNTs can also be used as potential nanoscale building blocks to construct three-dimensional (3-D) architecture ensembles on an electrode surface that are not readily available with other materials.12,13 The separated single nanotube and/or small bundle of the CNTs contained in such 3-D architecture could behave like many * Corresponding author. E-mail:
[email protected]. † Tsinghua University. ‡ University of Science and Technology of China.
individual nanoelectrodes. These electrodes, once being rationally designed, could be used to facilitate direct electron transfer of the proteins immobilized on them.14 Despite the fact that CNTs-based biosensors possess many advantages, there are still problems not being solved. For example, it is noticeable that activities of proteins or enzymes are often reduced when they are directly bound to the CNTs. It has been reported that the specific activity of soybean peroxidase (SBP) absorbed onto CNTs was about 40% of the activity of the native proteins in aqueous buffer, while the horseradish peroxidase remained only 33% of that.15 On the other hand, the direct electrochemical faradic responses achieved for the metalloproteins bound directly at CNTs are relatively weak.16 The enhanced faradic responses are of great importance in voltammetric investigations of interfacial electron transfer for metalloproteins at the CNTs electrode, and it is also highly desired for development of the CNTs-based bioelectronic devices. Furthermore, the proteins immobilized on CNTs via methods such as physical adsorption or entrapment tended to leach with time, thus making the CNTsbased biosensor unstable.16 Deoxyribonucleic acid (DNA), which is a well-known natural biocompatible macromolecule, has now gained increasing attention in the various biotechnology fields such as biosensor, bioimplant, and so forth.17-19 Recently, attention has also been paid to the important finding that the double-stranded DNA (dsDNA) can greatly affect the behavior of protein binding with it.20 Significantly, the activities of metalloproteins such as hemoglobin (Hb) were greatly enhanced by being immobilized on the surface of the solid matrix together with ds-DNA.21 Moreover, DNA is also a conductive natural polymer, whose stacked base pairs can be considered as a system of π-electrons connected to transfer electrons so that efficient electron migration within the DNA duplex is possible over a distance up to
10.1021/jp071551f CCC: $37.00 © 2007 American Chemical Society Published on Web 05/27/2007
8656 J. Phys. Chem. C, Vol. 111, No. 24, 2007 40 Å.22-24 In addition, DNA was often used to fabricate varieties of coating materials by electrostatic self-assembly, which was attributed to the negative charges originating from phosphate residues along the polynucleotide chain.25-27 On the basis of such an assembly method, hybrid nanostructures composed of DNA and various nanomaterials have become a subject of considerable interest for developing novel biomaterials.28 In this work, we fabricated hybrid nanocomposites based on the electrostatic assembly of Hb with MWNT and ds-DNA under mild conditions, in which Hb was sandwiched between MWNTs and ds-DNA layers. The unique sandwich-like nanostructures not only provided a favorable microenvironment in which to keep the bioactivity of Hb but also prevented leakage of the bound protein. Meanwhile, the DNA-Hb-MWNTs film preserved the 3-D featured structure of MWNTs assembled on the electrode, and it was found to possess good electron-transfer properties for the proteins with enhanced bioelectrochemical catalytic activities and faradic responses. The modified electrode also displayed fast response time, good sensitivity, wide linear range, and excellent stability to the detection of hydrogen peroxide. 2. Experimental Materials. Native double-stranded DNA (ds-DNA) from calf thymus and hemoglobin (Hb, MW 66 000) were purchased from Sigma Chemical Co. and used as received. Multiwall carbon nanotubes (MWNTs) were purchased from Shenzhen Nanotech Port Co. Ltd. H2O2 was purchased from Beijing Chemical Engineering Plant. All reagents were of the highest grade available and used without further purification. Deionized double-distilled water was used for making all the solutions (18 MΩ cm-1). Oxidation of MWNTs. MWNTs were purified and oxidized as reported previously.24 Typically, a 250 mg quantity of commercial MWNTs was refluxed in 500 mL of 2 M HNO3 for 2 days. The purified MWNTs were further oxidized by treatment with 60 mL of 1:3 HNO3/H2SO4 mixtures for 6 h in an ultrasonic bath. The suspension was diluted 10 times with water after removal of the clear solution over the precipitates. After the solution was filtered and further washed to neutral pH by a large amount of water, the resulting sample was dried in a vacuum for about 8 h. The oxidized MWNTs as prepared are water-soluble and stored as aqueous solutions of 2 mg/mL. Preparation of Hb-MWNTs Nanocomposite. A 4 mL quantity of 2 mg/mL Hb solution at pH 6.0 was added to 4 mL of 2 mg/mL MWNTs aqueous suspension. The mixture was stirred for 1 h and then kept at 4 °C for 12 h with occasional shaking. Subsequently, the mixture was centrifuged at 9000 rpm for 15 min to remove the supernatant. A quantity of 2 mL of deionized water was then added and the MWNTs were redispersed by gentle shaking. The centrifugation/wash/redispersion cycle was repeated three times to ensure removal of the free Hb or unadsorption Hb from MWNTs surface. The resulting Hb-MWNTs composites were then dispersed in 4 mL of pH 6.0 buffers and divided into two equal pieces for the further preparation of the Hb-MWNTs/GC electrode and DNA-HbMWNTs nanocomposites. Preparation of DNA-Hb-MWNTs Nanocomposite. A quantity of 2 mL of 0.5 mg/mL ds-DNA solution was added to 2 mL of Hb-MWNTs solution under vigorous stirring for 30 min. The resulting mixture was then centrifugated at 12 000 rpm for 10 min. Using the above procedure, the residue was washed 3 times with deionized water to yield the resulting DNA-HbMWNTs. Subsequently, the DNA-Hb-MWNTs was redispersed in 2 mL of pH 6.0 buffers and stored in a refrigerator at 4 °C.
Zhang et al. SCHEME 1: Assembly Procedure of DNA-Hb-MWNTs
Preparation of Hb-MWNTs/GC and DNA-Hb-MWNTs/ GC Electrodes. Prior to use, a GC electrode with a diameter of 3 mm was polished on a polishing cloth with 1.0, 0.3, and 0.05 µm alumina powder, respectively, and rinsed with deionized water followed by sonicating in acetone, ethanol, and deionized water successively. Then, the electrode was allowed to dry at room temperature. The modified electrode was prepared by a simple casting method. Typically, 5 µL of Hb-MWNTs and DNA-Hb-MWNTs dispersion were cast onto the GC electrode, respectively. A beaker was covered over the electrodes so that water can evaporate slowly in air and a uniform film electrode can be formed. Then dried Hb-MWNTs/GC and DNAHb-MWNTs/GC electrodes were stored at 4 °C in a refrigerator when not in use. Instrument. Electrochemical measurements were performed at room temperature using a CHI 660 workstation (CH Instruments, Inc., Austin, TX). The measurements were based on a three-electrode system with the as-prepared enzyme electrode as the working electrode, a platinum wire as the counter electrode, and a saturated Ag/AgCl electrode as the reference electrode. Without special statement, 0.1 M pH 6.0 PBS was used as the electrolyte solution in all experiments. The buffer solution was purged with highly purified nitrogen for at least 30 min, and a nitrogen atmosphere environment was maintained during all electrochemical measurements. UV-vis experiments were performed with a UV-2100S spectrophotometer (Shimadzu). The FTIR spectra of samples in KBr pellets were recorded on a Perkin-Elmer instrument. The morphologies of the as-prepared samples were observed utilizing a Hitachi model H-800 transmission electron microscope (TEM) opened at an accelerating voltage of 100 kV and JSM7401 scan electron micrographs (SEM). 3. Results and Discussion Self-assembly technology is an efficient method to fabricate hybrid nanostructure materials due to its simplicity of procedure, wide choice of materials, and precise control of the composition.29,30 The resulting composites with multifunctional and improved properties have been widely used in various fields.31,32 Scheme 1 depicts the idealized scheme for the design and construction of DNA-Hb-MWNTs nanocomposites with sandwich-like structures. The sonication-induced acid oxidation process used for purifying and cutting MWNTs led to carboxylate groups on the MWNTs surface, resulting in nanotubes that were negatively charged in neutral aqueous solution. Hb is an amphoteric protein (pH value at the isoelectric point is 7.0), which has a net positively charged surface at pH below its isoelectric point. Then when two oppositely charged composites
DNA-Hemoglobin-MWNTs Hybrid Material
Figure 1. UV-vis absorption spectra of Hb (a), ds-DNA (b), MWNTs (c), Hb-MWNTs (d), and DNA-Hb-MWNTs (e) in buffer of pH 6.80.
were mixed together at buffers of pH 6.0, the protein molecule adsorbed onto the surface of MWNTs to form Hb-MWNTs with core-shell structure. At the same time, charge overcompensation occurred, and thus the resulting Hb-MWNTs possessed positive charge. In contrast, DNA contains negative charges originating from phosphate residues along the polynucleotide chain. Therefore, after DNA was added to a solution of Hb-WMNTs, it could further assemble electrostatically on the surface of Hb-WMNTs to form the final DNA-Hb-MWNTs composites. Such hybrid composites could be visualized as a three-layered entity, where the outer sphere was the biocompatible DNA layer, the middle sphere was the Hb molecules attached to the surface of WMNTs, and the inner core was WMNTs which also acted as the skeleton of the assembly system. As shown in the scheme, the electrostatic interaction played an important role during the assembly procession. It was the driving force for the construction of the sandwich-like structure, and also made the protein immobilize into the sandwich structure under mild conditions. It is known that the mild conditions required for the immobilization process are very important to the retention of the protein native structure and activity. The self-assembly process was also monitored by UV-vis absorption spectroscopy as shown in Figure 1. The UV-vis spectrum of the pristine MWNTs (curve c in Figure 1) dispersed in aqueous solution exhibits a featureless absorption, whereas the spectra of free Hb (curve a in Figure 1) and DNA (curve b in Figure 1) in aqueous solution displayed a strong absorbance at 407 and 260 nm, respectively. After the MWNTs were oxidized and electrostatically interacted with Hb, the Soret band of Hb at 407 nm was found in the spectrum of Hb-MWNTs (curve d in Figure 1), thus indicating that Hb had been adsorbed successfully on MWNTs. Then Hb-MWNTs were treated with DNA, and it is evident that a new peak further appeared at 260 nm in the spectrum of the final DNA-Hb-MWNTs sample (curve e in Figure 1), which was due to the alternative absorption of DNA on Hb-MWNTs. Because the immobilization of proteins on a solid surface can result in the loss of native conformation to a significant extent or denaturation in some cases, spectroscopic studies such as UV-vis absorption are used to monitor possible structural changes of proteins. The absorption spectra of the free Hb, HbMWNTs, and DNA-Hb-MWNTs shown in Figure 1 indicate that the position of the Hb Soret band at 407 nm was unaffected during the multi-assembly steps, suggesting that the protein molecules were associated with other components without denaturation. FTIR absorption is also used to monitor possible structural changes of proteins because the vibrational bands of the amide groups in proteins provide sensitive signatures of protein secondary structure. As shown in Figure 2, the amide I
J. Phys. Chem. C, Vol. 111, No. 24, 2007 8657
Figure 2. FTIR spectra of Hb (a), DNA (b), Hb-MWNTs (c), and DNA-Hb-MWNTs (d). The amide I and II bands of free Hb, Hb-MWNTs, and DNA-Hb-MWNTs in FTIR spectra are located at 1658 and 1546 cm-1, respectively.
and II bands of Hb-MWNTs and DNA-Hb-MWNTs located at 1658 and 1546 cm-1 were all essentially the same as those of the native proteins, suggesting that the secondary structures of the bound proteins were undisturbed. Furthermore, the amide I band of DNA-Hb-MWNTs (curve d in Figure 2) was broader than that of the native proteins, which was due to the overlap of the base pairing band of DNA at 1700 cm-1 and the amide I band of Hb at 1658 cm-1. Such results further demonstrated that both the DNA and Hb were immobilized on MWNTs. The structural and morphological characters of DNA-HbMWNTs are obtained by TEM and SEM analyses. Figure 3 presents the typical TEM images of the oxidized MWNTs and DNA-Hb-MWNTs. It is noticeable that these two kinds of nanotubes were completely different, especially the edge of the sidewalls. As shown, the oxidized MWNTs (Figure 3) used here were more than 1 mm in length and about 20-50 nm in diameter with hollow tube structure and smooth sidewalls. By comparison, a coating film could be clearly observed around the outer face of oxidized MWNTs for DNA-Hb-MWNTs (Figure 3), which indicated the presence of biomolecules wrapped around the sidewall and bottom of MWNTs. SEM images showed the surface morphology of MWNTs and DNA-Hb-MWNTs films. Figure 4 shows that the MWNTs were cross-linked with each other with an average separation of about 100 to 200 nm, thus forming a highly porous 3-D architecture. Compared with other electrode material, such a porous structure is an important feature for the MWNTs electrode. The single bundle and/or small bundles of the nanotubes in the 3-D porous architecture essentially behave like individual nanoelectrodes, which not only provided a large specific surface but also facilitated direct electron transfer of the proteins, even of those far from the substrate electrode.13 As shown in Figure 4, it is obvious that the DNA-Hb-MWNTs film also exhibited a similar porous structure, demonstrating clearly that the modification of MWNTs did not change their 3-D structures on the electrode. Figure 5 shows typical cyclic voltammograms of different electrodes at 0.2 V s-1 in 0.1 M pH 6.0 PBS solution. No obvious redox peaks were observed at the bare MWNTs modified electrode (curve a in Figure 5) and the bare DNAmodified electrode (curve b in Figure 5), which showed that MWNTs and DNA were not electroactive in the potential range. After Hb assembled on the MWNTs, a pair of well-defined redox peaks were observed at the Hb-MWNTs/GC electrode (curve c in Figure 5) with the formal peak potential (E1/2) of -0.298 V, which could be ascribed to the direct electron transfer of the immobilized Hb on the MWNTs for the Hb-FeIII/FeII redox couple. The observed cyclic voltammetric responses demonstrated that MWNTs enhanced direct electron transfer
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Zhang et al.
Figure 3. TEM images of the multiwalled carbon nanotubes (a) and DNA-Hb-MWNTs (b).
Figure 4. SEM images of the multiwalled carbon nanotubes film (a) and DNA-Hb-MWNTs films (b).
Figure 5. Cyclic voltammograms of MWNTs/GC (a), DNA/GC (b), Hb-MWNTs/ GC (c), and DNA-Hb-MWNTs/GC (d) electrodes in 0.1 M PBS (pH 6.0). Scan rate, 0.2 V s-1.
between Hb and the underlying electrode; this result was reported previously.33 As shown in Figure 5d, a pair of welldefined redox peaks was also observed at E1/2 of -0.298 V for a DNA-Hb-MWNTs/GC electrode. Because DNA was not electroactive in this potential range, the redox peaks were apparently attributed to a quasi-reversible electron-transfer process of the Hb-FeIII/FeII which was immobilized between MWNTs and DNA. Compared with the redox peak currents obtained at the Hb-MWNTs/GC electrode (curve c in Figure 5), remarkably larger currents were obtained at the DNA-HbMWNTs/GC electrode. The enhanced faradic current response for immobilized Hb at the DNA-Hb-MWNTs/GC electrode
(6.9 µA) was about 4 times higher than that at the Hb-MWNTs/ GC electrode (1.7 µA). The reduction and oxidation peak currents increased linearly with scan rates, and the integration of reduction peaks gave nearly constant charge values (Q) with different scan rate, indicating that the redox process of the immobilized Hb in the sandwich-like nanostructure was a surface-confined process (Figure S1). According to Faraday’s law, Q ) nFAΓ* (where F is the Faraday constant and Γ* represents the surface concentration of electroactive Hb, Q can be calculated by integrating the reduction peak of Hb, n stands for the number of electrons transferred, and A represents the area of the electrode surface, here using the geometric area of the GC electrode (0.07 cm2)). Thus, the surface concentration of electroactive Hb (Γ*) at the DNA-Hb-MWNTs/GC electrode was calculated to be 1.18 × 10-9 mol cm-2, which was about 4 times higher than that at the Hb-MWNTs/GC electrode (2.86 × 10-10 mol cm-2) and was also larger than the theoretical monolayer value (1.89 × 10-11 mol cm-2). Therefore, the MWNTs-based electrode for immobilization of Hb with DNA showed a much higher surface concentration of electroactive Hb than that in the absence of DNA, which indicates that 3-D sandwich-like nanostructures provided multilayered immobilization for Hb in the electron-transfer process. The striking distinction of these voltammetric responses between these two electrodes demonstrated that the portion of the electroactive proteins was greatly improved after Hb was sandwiched between MWNTs and the the DNA outer layer. It is noticeable that the
DNA-Hemoglobin-MWNTs Hybrid Material
J. Phys. Chem. C, Vol. 111, No. 24, 2007 8659
Figure 6. Cyclic voltammograms of DNA-Hb-MWNTs/GC electrode in 0.1 M PBS buffer solution (pH 6.0) with 0 µM (a), 105 µM (b), and 175 µM (c) H2O2. Scan rate, 0.2 V s-1.
Hb-MWNTs and DNA-Hb-MWNTs materials and the corresponding modified electrodes were fabricated under almost identical conditions, the only difference being that a DNA layer was further absorbed on the immobilized Hb. On this basis, we speculated that the observed increased ratio of electroactive Hb and the remarkably enhanced faradic current responses at the DNA-Hb-MWNTs/GC electrode were associated with the DNA outer layer. The reason for the facilitation of the direct electron transfer of Hb was possibly due to the good biocompatibility of the hybrid material and the favorable orientation of the immobilized Hb provided by the outer layer of DNA. In conclusion, the direct electron transfer behavior of the immobilized metalloproteins on WMNTs was greatly affected by the DNA outer layer bound with them, and such enhanced faradic responses of proteins are attractive for the development of enzymatic biosensors or bioreactors with improved performance.14 As is well-known, the monitoring of hydrogen peroxide is of great importance in medicine, environmental control, and industry. To check the bioelectrocatalytic activity of the DNAHb-MWNTs/GC electrode to the reduction of H2O2, cyclic voltammetric experiments were performed. When H2O2 was added to a pH 6.0 PBS solution, an obvious increase of the reduction peak current at about -0.30 V was observed, accompanied by the decrease of the oxidation peak current and an increase of reduction peak current with increasing H2O2 concentration (see Figure 6). These results indicated that Hb immobilized in the DNA-Hb-MWNTs naonocomposites kept its bioelectrocatalytic activity. In order to compare the bioelectrocatalytic performance of the Hb-MWNTs/GC electrode with that of the DNA/Hb/ MWNTs/GC electrode, the electrocatalytic reduction of H2O2 at both modified electrodes was studied by chronoamperometry. The catalytic reduction currents were monitored when aliquots of H2O2 were added. The typical current-time curves for the Hb-MWNTs/GC electrode and the DNA-Hb-MWNTs/GC electrode are shown in Figure 7A. Both of these two modified electrodes displayed fast response time (t95% < 6 s), which was mainly due to the easy diffusion of H2O2 in the open 3-D architecture of the nanocomposites on the electrodes. However, the DNA-Hb-MWNTs/GC electrode exhibited larger stepped growth of reduction currents than that of the Hb-MWNTs/GC electrode. As shown in Figure 7B, the amperometric currents obtained at these two electrodes all had a good linear relationship with the concentration of H2O2. Comparing curve A and curve B, the linear range of the DNA-Hb-MWNTs/GC electrode and the Hb-MWNTs/GC electrode to H2O2 was 35-1400 and 35945 µM, respectively. From the slope of the curve, the sensitivity
Figure 7. (A) Chronoamperometric curves at the constant potential of -0.1 V (vs Ag/AgCl) in pH 6.0 buffers with injection of H2O2 every 30 s for Hb-MWNT/GC (a), DNA-Hb-MWNTs/GC (b) electrode. (B) Plot of the electrocatalytic current (Icat) versus H2O2 concentration for Hb-MWNT/GC (a), DNA-Hb-MWNTs/GC (b) electrode in 0.1 M PBS (pH 6.0) buffer solution.
of the DNA-Hb-MWNTs/GC electrode and the Hb-MWNTs/ GC electrode is calculated to be 9.0 and 6.9 mA cm-2 M-1, respectively. Therefore, it is obvious that the former one attained a wider linear range and higher sensitivity, indicating that the DNA/Hb/MWNTs/GC electrode exhibited better electrocatalytic performance to H2O2 than the Hb-MWNTs/GC electrode. Since these two modified electrodes have the same protein loading, the wider linear range and higher sensitivity of the DNA-HbMWNTs/GC electrode may be attributed to the enhanced direct electron transfer of Hb in the hybrid material and the remaining 3-D structures of DNA-Hb-MWNTs film on the electrode. The stability of the DNA-Hb-MWNTs/GC electrode was also examined by CV under “wet” and “dry” conditions, respectively. Under the “wet” condition, the modified electrode was stored in pH 6.0 buffers all the time and CVs were performed after a period of storage time. The decrease of the cathodic peak current was less than 2.5% after 5 h, demonstrating that the DNA-HbMWNTs/GC electrode was stable in buffer solution. Under the “dry” condition, the modified electrode was stored in air at 4 °C. The DNA-Hb-MWNTs/GC electrode still retained 95% of its initial response current after 15 days storage. Accordingly, the DNA-Hb-MWNTs electrode exhibited good stability under these two different conditions, which can be attributed to the excellent biocompatibility of DNA-Hb-MWNTs hybrid material that offered a favorable microenvironment to the immobilized Hb. On the other hand, the outer layer of DNA could assist MWNTs and Hb to attach to the electrode more stably because of the excellent film-forming ability of DNA, and the leakage of the immobilized protein can also be efficiently prevented due to the confinement of the DNA shell at the same time.
8660 J. Phys. Chem. C, Vol. 111, No. 24, 2007 4. Conclusion In summary, a novel hybrid biomaterial with sandwich-like structure has been successfully fabricated by the alternative assembly of metalloproteins and DNA on MWNTs. The resulting DNA-Hb-MWNTs not only preserved the feature 3-D structure of MWNTs on the electrode, but also possessed good electron-transfer properties for the proteins with enhanced bioelectrochemical catalytic activities and faradic responses. Compared with the Hb-MWNTs/GC electrode, the prepared DNA-Hb-MWNTs/GC electrode displayed better ability to keep the bioelectroactivity of Hb and exhibited high electrocatalytic performance to H2O2 with fast response, wide linear range, good sensitivity and excellent stability. Such an avenue, which integrated MWNTs, metalloproteins and DNA into an organized multicomponents platform, may open up a new route for the fabrication of biosensors, biofuel cells and other bioelectrochemical devices. Acknowledgment. This work was financially supported in part by the NNSFC (20435010, 20675044). Supporting Information Available: Cyclic voltammograms of the DNA-Hb-MWNTs/GC electrode. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Armstrongh, F.; Hill, A. O.; Walton, N. J. Acc. Chem. Res. 1988, 21, 407. (2) Zhou, Y.; Hu, N.; Zeng, Y.; Rusling, J. F. Langmuir 2002, 18, 211. (3) Topoglidis, E.; Astuti, Y.; Duriaux, F.; Gratzel, M.; Durrant, J. R. Langmuir 2003, 19, 6894. (4) Zhao, G.; Feng, J. J.; Xu, J. J.; Chen, H. Y. Electrochem. Commun. 2005, 7, 724. (5) Zhang, Z.; Chouchane, S.; Magliozzo, R. S.; Rusling, J. F. Anal. Chem. 2002, 74, 163. (6) Ajayan, P. M. Chem. ReV. 1999, 99, 1787. (7) Baughman, R. H.; Zakhidov, A. A.; Heer, W. A. D. Science 2002, 297, 787. (8) Yang, M.; Yang, Y.; Yang, H.; Shen, G.; Yu, R. Biomaterials 2006, 27, 246.
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